DE19605657A1 - Proinsulin derivative and process for the production of human insulin - Google Patents

Abstract

There is described a human proinsulin derivative of the formula: human insulin B chain - Z - human insulin A chain wherein Z is a polypeptide linker of the formula: U-U-X-P-J-(X')n-U-U in which: U is arginyl or lysyl; X and X' are amino acid radicals; P is prolyl; J is glycyl, arginyl or lysyl; n is 0 or an integer of from 1 to 5. There is also described a DNA sequence encoding the proinsulin derivative, which may be inserted into an expression vector. The vector is used to transform a microorganism, preferably Escherichia coli . Human insulin is prepared from the proinsulin derivative by enzymatic hydrolysis.

Description

The present invention relates to human proinsulin derivative and a process for producing human sulin using an expression vector which is the one DNA encoding human proinsulin derivative. More specific it concerns a human proinsulin derivative which instead of C-peptide is a short peptide sequence between the A and B chains of insulin; an expression vector, the one that DNA encoding human proinsulin derivative; one with the expression vector transformed microorganism and a Process for the production of human insulin by Cultivation of the microorganism in a suitable medium.

Insulin is a polypeptide hormone produced by the Pan kreas is released and at the control of blood sugar levels participates. It consists of two peptide chains, i. H. the A and the B chain attached to their cysteine residues via disulfide bridges are connected and it is processed by proteolytic processing Proinsulin produced in the pancreatic β-cells (insulin: Molecular Biology and Pathology, ed. Ashcroft, F.M. & Ashcroft, S.J.H., IRL Press, Oxford, 1992). Commercial is human Insulin is either produced by an enzymatic process with an alanine residue at position 30 of the B chain of swine insulin by a transpeptidation reaction  Use of trypsin was replaced by a threonine residue (Markussen, J., Proceedings 1st International Symposium "Neue Insuline ", pp. 38-44 (1982), Ed. Petersen, K.G. et al., Freiburger Graphische Betriebe, Freiburg); or by a Procedure using genetically modified E. coli (Chance, R.E. et al., Peptides: Synthesis-Structure-Function, S. 721-728 (1981), in Proceedings of the Seventh American Peptide Symposium, Ed. Rich, D.H. & Gross, E., Pierce Chemical Co., Rockford, IL, U.S.A .; and Frank, 3. H. et al., Peptides: Synthesis-Structure-Function, pp. 729-738 (1981), in Proceedings of the Seventh American Peptide Symposium, Ed. Rich, D.H. & Gross, E., Pierce Chemical Co., Rockford, IL, U.S.A.) or Saccharomyces cerevisiae (Thim, L. et al., Proc. Natl. Acad. Sci. U.S.A., 83, pp. 6766-6770 (1986); and Markussen, J. et al., Protein Engineering, 1, pp. 205-213 (1987)). Because the enzymatic Process for the production of human insulin Pig insulin is limited because of its high cost recent investigations on processes for the production of human insulin by genetic engineering techniques.

Chance et al. have reported a process in which In sulin is produced in that both the A- and the B- Chain of insulin in the form of a fusion protein by growing E. coli is produced which comprises a vector which is a the Contains DNA encoding fusion protein; Splitting the merger proteins with cyanogen bromide, which maintains the A and B chains will; Sulfonation of the A and B chains, causing sulfonation Chains are obtained; Implement the sulfonated B chain with an excess of the sulfonated A chain and then purifying the  resulting product, which gives insulin (chance to RE. et al., supra). However, this procedure points to the extent Disadvantages that it is cumbersome, two Fermentationsver drive to operate, and that the reaction of the sulfonated A and B chains only give a low yield of insulin, which is what Makes the process itself impractical.

Frank et al. have of a process for the production of In sulin reports that it includes the following steps: Manufacture of proinsulin in the form of a fusion protein by growing of E. coli, which has a vector that is a fusion contains protein coding DNA; Cutting the fusion protein with Cyanogen bromide to give proinsulin; Sulfonation of Proinsulin and separation of the sulfonated proinsulin; Fold back of sulfonated proinsulin to form the correct ones Disulfide bonds; Treat the refolded proinsulin with Trypsin and carboxypeptidase 3; and then cleaning the obtained one Product, whereby insulin is obtained (Frank et al., Supra). The yield of refolded proinsulin with the correct ones However, disulfide bonds decrease sharply when the concentration of proinsulin increases. This fact is due to misfolding and causes some degree of polymerization, so the process has the disadvantage that during the extraction of Proinsulin carried out labor-intensive cleaning steps Need to become.

Thim et al. have reported a process for producing insulin in Saccharomyces cerevisiae which comprises the steps of:
Prepare a single chain insulin analog with a particular amino acid sequence by growing Saccharomyces cerevisiae cells and isolating the insulin therefrom by a series of steps, i. H. cleaning, enzyme conversion, acid hydrolysis and further cleaning (Thim, L et al, supra). Although this method is advantageous in that the purification steps are relatively simple and no refolding process is necessary, it also results in only a low insulin yield due to the low degree of expression of a yeast system compared to E. coli.

The role of the C peptide in folding proinsulin is not exactly known. A biochemical textbook describes that the C peptide is necessary for the folding process (Biochemistry, 3rd edition, p. 41 (1988), Freeman), other Un However, tests have shown that approximately 30 to 50% are correct folded insulin can be obtained if the A and B Chains alone in the absence of the C peptide in very little Concentrations are used (Katsoyannis, P.G. et al., Biochemistry, 6, pp. 2642-2635 (1967); and Chance, R.E. et al., supra). It has also been shown that when using a Peptide in which the A and B chains are directly linked are a very high yield of correctly folded product can be kept, which is comparable to the yield that can be achieved using proinsulin (Steiner, D. F. & Clark, J.L., Proc. Natl. Acad. Sci. U.S.A., 60, pp. 622-629 (1968); and Varandani, P.T. & Nafz, M.A., Arch. Biochem. Biophys., 141, pp. 533-537 (1970)). These results suggest that the role of the C peptide is simply to control the A and B Bring chains close together so the folding process simplify.

According to the known three-dimensional structures of insulins in the Brookhaven database is the distance between the α- Carbon atom at the C-terminus of the B chain (Thr-30) and the α- Carbon atom at the N-terminus of the A chain (Gly-1) about 5 to 11  Å, which is a suitable distance for the insertion of a β- Represents loop structure. It's been a long time knows that certain amino acid sequences are highly likely are part of a loop conformation in proteins (Chou, P.Y. & Fasman, G.O., J. Mol. Biol., 115, 135-175 (1977)), and it has recently been shown that this also applies to peptides in aqueous solution (Dyson, H. J. et al., J Mol. Biol., 201, 161-200 (1988); and Shin, H.C. et al., Biochemistry, 32, 6348-6355 (1993)). It was found that Proline in the second position and glycine in the third position result in the highest β-loop population, and an extensive one Examination showed that the type of amino acids at position 4 affects the stability of the β-loop in the trans position, and a deprotonated Asp 4 side chain is preferred (Wright, P.E. et al., Biochemistry, 27, 7167-7175 (1988); and Dyson, H.J. et al., Supra).

β loops are likely places to start one Protein folding and being determined by short interactions limit the available one for the polypeptide chain Conformational space, and they can use it to control after of the following folds contribute to distant parts of the Bring the polypeptide chain together (Zimmerman, S. S. & Scheraga, H. A., Proc. Natl. Acad. Sci. U.S.A., 74, 4126-4129 (1977); and Wright, P.E. et al., Supra). It is also known that these β- Grinding plays an important role in enzymatic cleavages play.

Trypsin is a typical serine protease and hydrolyzes one Protein or peptide at the carboxy terminus of an arginine or lysine residues (Enzymes, pp. 261-262 (1979), ed. Dixon, M. & Webb, E.C., Longman Group Ltd., London). Especially arrives  a dibasic site where two successive argi nin or lysine residues exist, easily hydrolysis, and it is known that this hydrolysis is most likely to take place there, where the dibasic site is in or next to a β-loop structure is arranged (Rholam, Met al., FEBS Lett., 207, 1-6 (1986)).

As described above, there is a need for methods with high yields for the production of human insulin in one Microorganism, and the inventors of the present invention have undertaken an improved manufacturing process to develop insulin and have a procedure with high Yield found, being a proinsulin derivative with a low Molecular weight and an easily hydrolyzable β- Loop structure is used.

Accordingly, it is an object of the present invention to provide a human proinsulin derivative that has a high folding yield and is easily hydrolyzable, and one of these derivatives to provide coding DNA.

It is another object of the present invention to provide a Expression vector for the expression of human proinsulin to provide, which comprises the DNA.

It is another object of the present invention to provide one with the expression vector transformed microorganism be to sit down.

It is still another object of the present invention Process for the preparation of human insulin to deliver the cultivation of the transformed microorganism in an appropriate medium whereby human proinsulin  is produced, the separation of human proinsulin thereof and producing human insulin from the proinsulin includes.

The foregoing and other tasks and features of the present ing invention are by the following description of Er together with the accompanying drawings to whom:

Fig. 1 represents a schematic diagram of the preparation of the plasmids pT7-T1-T2 and pT7;

Figure 2 shows a schematic diagram of the production of plasmids pT1-hPI and pT2-hPI;

Figure 3 is a schematic diagram of the production of plasmids pT1-M1PI, pT1-M2PI, pT1-M3PI and pT1-M4PI;

Figure 4 is a schematic diagram of the production of plasmids pT2-M1PI, pT2-M2PI, pT2-M3PI and pT2-M4PI;

Figure 5 shows the HPLC chromatograms of refolded M1PI, M2PI, M3PI, M4PI and hPI; and

Figure 6 shows the HPLC chromatograms of insulins formed by hydrolysis of refolded miniproinsulins (MiPIs) and hPI.

All references cited herein are hereby incorporated by reference included on their entire content.

According to the present invention, a human proinsu Linderivat of formula (I) provided below as "Miniproinsulin" is referred to in which a peptide is derived from there is a maximum of 12 amino acids and a fixed β-loop structure forms, instead of the 35 amino acids C-peptide inserted between the A and B chains of human insulin  becomes:

B-chain-Z-A-chain (I)

where Z is a peptide of the formula UUXPJ- (X ′) nUU,
U is an arginine or lysine residue;
X and X 'are independently amino acid residues and
(X ′) nn can be different or the same amino acid residues;
P is a proline residue;
J is a glycine, arginine or lysine residue; and
n is 0 or an integer from 1 to 5.

Preferred miniproinsulins are those in which X is an alanine, Is tyrosine, histidine or glycine residue; X ′ an aspartic acid, Is valine, arginine or glycine residue; and n is 0 or 2. Exemplary preferred miniproinsulins are those in which Z Arg-Arg-Ala-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO: 1); Arg-Arg-Tyr- Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO: 2); Arg-Arg-His-Pro-Gly-Asp- Val-Lys-Arg (SEQ ID NO: 3) or Arg-Arg-Gly-Pro-Gly-Lys-Arg (SEQ ID NR: 4).

The mini proinsulin of the present invention can be used alone or in Form of a fusion protein, wherein it with a Fusion partner protein, preferably a beta structure forming protein is fused.

Exemplary fusion partner proteins described in the present Invention can be used encompass the entire human tumor necrosis factor (TNF) or parts thereof with the amino acid sequence shown below (SEQ ID NO: 5), the ge whole mutated TNF or parts thereof, in which some of the Ami noacids in SEQ ID NO: 5 have been replaced by other amino acids and those polypeptides in which 1 to 7 are preferably 7,  Amino acids from which TNF's N-terminus has been removed:

A human TNF or a mutant TNF encoding DNA can be chemical using a DNA synthesizer or via a polymerase chain reaction ("PCR") (Saiki, K.K. et al., Science, 230, pp. 1350-1354 (1985)), wherein human TNF cDNA as a template and suitable, chemically produced posed primer can be used.

The DNA encoding part or all of the mutant TNF can e.g. B. can be produced by the following steps: Win of human TNF DNA by PCR from a U937 cell line human monocyte cDNA library (Clontech, USA.); and performing another PCR using the TNF DNA as a template and from chemically produced primers, i.e. H. a sense primer that is a recognition site for a Restriction enzyme and modified nucleotide sequences on his  5'-end, and an anti-sense primer, the one Detection site for a restriction enzyme at its 5'-end having.

The mini proinsulin of the present invention can chemically a peptide synthesizer according to a conventional method or using a conventional genetic engineering method in a microorganism.

For the production of a miniproinsulin in a microorganism becomes a compatible host cell, e.g. B. an E. coli or saccha romyces cerevisiae cell with a transfor expression vector miert, the a DNA encoding the miniproinsulin alone, or contains a fused DNA that is from a fusion partner protein and the miniproinsulin existing fusion protein ko dated; and the transformed cell is ge under conditions breeds, which enables the expression of the miniproinsulin. Then the miniproinsulin produced is refolded by a Proteinase hydrolyzes and is used to obtain human insulin cleaned.

Accordingly, the present invention provides a method for Production of insulin provided the following Steps include: preparing a miniproinsulin encoding DNA; Introducing the DNA alone or together with one DNA encoding fusion partner protein into a suitable vector, La an expression vector for the expression of the miniproinsulin manufacture; Transform a host cell with the Ex pressure vector; Cultivate the resulting transformant under Conditions that allow expression of the miniproinsulin, Separating the Mini Proinsulin from the Culture and Making Insulin from the mini proinsulin.  

An expression vector system can be created by introducing the Mini proinsulin alone encoding DNA or by sequential Introduce the fusion partner protein and the miniproinsulin encoding DNAs are produced in a vector, the one suitable promoter, e.g. B. lac, trp, tac, pL, T3, T7, SP6, SV40 and contains 1 (pL / pR); or by ligating the miniproinsulin or DNA encoding the fusion protein with a DNA encoding a this contains promoters and a ribosome binding region, followed by inserting the ligated DNA into an appropriate one Plasmid, e.g. B. pBR322.

The expression vectors prepared above can be according to usual methods, e.g. B. Cohen's method as described in Proc. Natl. Acad. Sci. U.S.A., 69, 2110 (1972), in a suitable host cell, e.g. B. E. coli are introduced under Taking into account a number of well-known in the field known factors is selected, e.g. B. compatibility with selected vector, the ease, the desired polypeptide to gain, the polypeptide properties, the biological Security and the cost of transforming a transformed E. coli Cell is obtained.

Two such transformants were identified as E. coli BL21 (DE3 + pT1M1PI) and E. coli BL21 (DE3 + pT2M2PI) and on December 29, 1994 at the Korean Culture Center of Microorganisms (KCCM) (Address: Department of Food Engineering, College of Eng., Yonsei University, Sodaemun-gu, Seoul 120-749, Korea) with the Filing numbers KCCM-10059 or KCCM-10060 under the Terms of the Budapest Treaty on International Acknowledgment of deposit of microorganisms for the purposes deposited by patent proceedings.  

The transformed cells are grown under conditions which enable expression of the miniproinsulin.

The polypeptides produced in a host cell can be isolated and the desired miniproinsulin can be obtained through the combined use of conventional methods, e.g. B. the Cell destruction, centrifugation, sulfitolysis, dialysis, treatment be separated with cyanogen bromide and HPLC.

In particular, this can be in E. coli in the form of a fusion protein comprehensive inclusion body produced by miniproinsulin the following procedures are cleaned.

The cells in the culture are destroyed by ultrasound, and the inclusion bodies containing the fusion protein by Zen trifugation collected. The inclusion bodies are in a ge suitable solvents, e.g. B. 6M guanidine HCl dissolved; the -SH- Groups of the cysteine residues in the protein are removed by sulfitolysis transferred to -SSO₃ groups, and the proteins are then by Dialysis or gel filtration chromatography failed. The precipitated proteins are collected by centrifugation and washed several times with an aqueous solution. The resulting Protein is treated with cyanogen bromide and the product obtained purified by HPLC, whereby the miniproinsulin is obtained.

The miniproinsulin is then refolded with β-mercap treated to ethanol, and the refolded mini proinsulin with trypsin and carboxypeptidase B, preferably in such amounts treated that weight ratios of trypsin: carboxypeptidase B: Miniproinsulin of 1: 5: 12,500 can be set so that Insulin is obtained.  

As described above, the present Er possible compared to the yield of human insulin the state of the art by expressing a human mini to improve proinsulin, instead of the bulky C-peptide in proinsulin a short peptide between the A and B chains of human insulin is introduced. The short peptide is there from only 7 to 12, preferably 9, amino acids, but it is able to form a firmer β-loop structure than the C- Peptide. The processes of refolding and hydrolysis can therefore with the miniproinsulin more efficiently than with the proinsulin.

The following examples are intended to further illustrate the present invention explain without limiting its scope.

The following for solid-in-solid, liquid-in-liquid and solid percentages given in liquid mixtures refer to a G / G, V / V or G / V basis, unless otherwise stated.

PCRs are in the examples according to the usual in Saiki, K.K. et al., supra, PCR methods described.

example 1 Preparation of the plasmid pT7-T150 <Step 1 <Production of TNF DNA

A polymerase chain reaction was used to obtain a TNF DNA (PCR) according to the method of Saiki, K.K. et al., Supra, below Use of a human monocyte cDNA library (Clontech. U.S.A.) made from a U937 cell line  as a template and from P1 primers with the following Nucleotide sequences performed:

Primer P1
Sinn Primer (SEQ ID NO: 6):

5′-GCCATACATA TGGTCAGATC ATCTTCTCGA ACC-3 ′
Antisense Primer (SEQ ID NO: 7)
3′-TGAAA CCCTAGTAAC GGGACACTAT TCCTAGGTGT-5 ′

<Step 2 < Production of a DNA for a mutated TNF containing plamids pT7-T150

In order to obtain a DNA for mutant TNF, a series of PCRs were carried out, using the TNF DNA produced in <step 1 <as a template and P2 and P3 primers with the following nucleotide sequences:
Primer P2 (which includes the desired nucleotide substitution)
Sinn Primer (SEQ ID NO: 8):

5′-GTGGTGCCAA TAGAGGGCCT GTTCCTCATC TAC-3 ′
Antisense Primer (SEQ ID NO: 9):

3′-cAC CACGGTTATC TCCCGGACAA GGAGTAGATG-5 ′
Primer P3 (complementary to the 5'- and 3'-ends of the TNF DNA)
Sinn Primer (SEQ ID NO: 10):

5 ′ -ATACATATGC CGAGTGACAA GCCTGTA-3 ′
Antisense Primer (SEQ ID NO: 11):

3′-A TGAAACCCTA GTAACGGGCC CCTAGGTACA-5 ′.

The first PCR was performed using the in <Step 1 < provided TNF DNA as a template, a P2 Sinn primer and a P3 Antisense primers were performed, and the second PCR was performed after same procedure as for the first PCR, except that uses a P2 antisense primer and a P3 sense primer were. The two resulting PCR products were mixed and then anneal.

Then the DNA for the mutant TNF was used by PCR using application of a P3 Sinn primer with an NdeI recognition site its 5'-end and a P3 antisense primer with SmaI- and BamHI- Detection sites amplified at its 3'-end. The resulting PCR product called T150 encodes a mutant TNF wherein 7 amino acids have been removed from the N-terminus of TNF and serine at position 52, tyrosine at position 56 and leucine at the 157th position of the TNF by isoleucine, Phenylalanine or arginine had been replaced.

T150 was digested with NdeI and BamHl and then using ligated from T4 DNA ligase to the Ndel / BamHI site of a vector, by digesting plasmid pT7-7 (Department of Biological Chemistry, Harvard Medical School) with NdeI and BamHl  had been. The resulting plasmid was called pT7-T150 designated.

Example 2 Preparation of the plasmid pT7-T1

A PCR was carried out using that prepared in <Step 1 < TNF DNA as a template, P3 as a sense primer and an anti-sense Primer with the following nucleotide sequence:

Antisense Primer (SEQ ID NO: 12):

3′-G ACATGGAGTA GATGAGGGCC CCTAGGTACA-5 ′

The resulting PCR product, called T1, encodes one mutant TNF, wherein 7 amino acids from the N-terminus of TNF had been removed, and serine at the 52nd position and glutamine replaced at the 61st position by isoleucine or arginine were.

T1 was digested with NdeI and BamHI and then ligated to the NdeI / BamHI site of a vector made by digesting plasmid pT7-7 with NdeI and BamHI using a T4 DNA ligase. The resulting plasmid was named pT7-T1 and its method of preparation is shown in Figure 1.

Example 3 Preparation of the plasmid pT7-T2

It became a synthetic Po encoding a polypeptide lynucleotide T2 produced. The polypeptide contains the 8th to 12th Amino acids of the N-terminus of TNF and also ten histidine residues,  that facilitate its cleaning. The following amino acid sequence is encoded in T2 (SEQ ID NO: 13):

H₂N-Pro Ser Asp Lys Pro His His His His His His His His His His Ser Ser-COOH

PCR was performed using primers T20-I, T20-II and T20-III performed in a ratio of 10: 1: 10, whereby a nucleotide comprising the T2 polynucleotide and NdeI and BamHI at their 5'- or 3'-ends has been:

T20-I (Sinn Primer; SEQ ID NO: 14):

5′-TATACATATG CCGAGTGACA AGCCT-3 ′
T20-II (Sinn Primer; SEQ ID NO: 15):

5′-AGTGACAAGC CTCATCATCA TCATCATCAT CATCATCATC ACAGCAG-3 ′
T20-III (antisense primer; SEQ ID NO: 16):

3'-TAGT AGTGTCGTCG CCTAGGTACA-5 ′

The resulting PCR product was digested with NdeI and BamHI and then ligated using a T4 DNA ligase to a plasmid pT7-7 treated with NdeI / BamHI. The resulting plasmid was named pT7-T2 and its method of production is shown in FIG. 1.

Example 4 Preparation of the plasmid pT150-hPI <Step 1 < Production of human proinsulin DNA

Based on the information about the amino acid sequence of human proinsulin (Ullrich, A. et al., Science, 612-615 (1980)) became a synthetic, human proinsulin code DNA that has the following nucleotide sequence (SEQ ID NO: 17) has, before using to a great extent in E. coli pulled codons to increase the rate of expression of the DNA increase:

In particular, oligonucleotides with the following nucleotide sequences for use in the production of a proinsulin gene were made using an ABI DNA Automatic Synthesizer (Model 392) using the solid phase phosphite synthesis method:
pTA (SEQ ID NO.18):

pTB (SEQ ID NO: 19):

pTC (SEQ ID NO: 20):

pTD (SEQ ID NO: 21):

pTE (SEQ ID NO: 22):

p31 (SEQ ID NO: 23): 5′-GTACCTGGTT TGCGGTGAAC GTGGT-3 ′
p112 (SEQ ID NO: 24): 3′-TAAC ATTGATCATT TTCGAAGCAT-5 ′
pBAM (SEQ ID NO: 25): 5′-GCAGGATCCA TGTTCGTTAA T-3 ′
pHIN (SEQ ID NO: 26): 3′-ATAA CATTGATCAT TTTCGAAACG-5 ′
p7l (SEQ ID NO: 27): 5′-GGTGCAGGCT CTCTGCAGCC GTTGG-3 ′
p72 (SEQ ID NO: 28): 3′-CCGAG AGACGTCGGC AACCGCGACC-5 ′

A DNA fragment was obtained by PCR using the Oli gonucleotides pTA and pTB as templates and the oligonucleotide p71  and p112 prepared as a primer and designated as a fragment. Another fragment called pCD fragment was also made using the same procedure using the Oligonucleotides pTC and pTD as templates and the oligonucleotides p31 and p72 prepared as primers. Equal amounts of the fragments pAB and pCD were mixed and then annealed. A third Fragment, which was designated fragment pABCD, was then prepared by PCR, the annealed oligonucleotide as Template and the oligonucleotides p31 and p112 used as primers were.

In addition, equal amounts of the pABCD fragment and the Oli gonucleotides pTE mixed and then annealed. It became one of those DNA encoding all human proinsulin by PCR prepared, the annealed oligonucleotide as a template and the oligonucleotides pBAM and pHIN were used as primers.

<Step 2 < Preparation of the plasmid pT150-hPI

The DNA for human proinsulin obtained in <step 1 < was digested with BamHI and HindIII, creating a double-stranded DNA with cohesive ends was made at the recognition sites contained for BamHI and HindIII at their 5'- and 3'-ends.

The plasmid pT7-T150 prepared in Example 1 was treated with BamHI and Hind III and then digested using T4 DNA ligase with BamHI / HindIII treated DNA for human Proinsulin obtained above ligated. That ent standing plasmid was called plasmid pT150-hPI.

Example 5 Preparation of the plasmid pT1-hPI

The hPI DNA obtained in <Step 1 <of Example 4 was also used BamHI and HindIII digested, creating a double-stranded DNA with cohesive ends was obtained, the recognition sites for BamHI and HindIII contained at their 5'- and 3'-ends.

The plasmid pT7-T1 prepared in Example 2 was digested with BamHI and HindIII and then ligated using T4 DNA ligase with the BamHI / HindIII treated hPI DNA obtained above. The resulting plasmid was named pTl-hPI and its production process is shown in Fig. 2.

Example 6 Preparation of the plasmid pT2-hPI

The hPI DNA obtained in <Step 1 <of Example 4 was also analyzed BamHI and HindIII digested, creating a double-stranded DNA with cohesive ends was obtained, the recognition sites for BamHI and HindIII contained at their 5'- and 3'-ends.

The plasmid pT7-T2 prepared in Example 3 was digested with BamHI and HindIII and then ligated using T4 DNA ligase to the BamHI / HindIII treated hPI DNA obtained above. The resulting plasmid was named pT2-hPI and its method of preparation is shown in Figure 2.

Example 7 Preparation of the plasmid pT1-MiPI

Mini proinsulins, each one between the A and B chains arranged, encoding a β-loop-forming peptide DNAs were prepared in a way that the second amino acid  of the peptide is a proline residue which is known to be a causes a fixed β-loop structure (Dyson, H. J. et al., J. Mol. Biol., 201, pp. 161-200 (1988)). The peptide is Arg-ArgAla-Pro Gly-Asp-Val-Lys-Arg (SEQ ID NO: 1) (M1PI); Arg-Arg-Tyr-Pro-Gly- Asp-Val-Lys-Arg (SEQ ID NO: 2) (M2PI); Arg-Arg-His-Pro-Gly-Asp- Val-Lys-Arg (SEQ ID NO: 3) (M3PI); or Arg-Arg-Gly-Pro-Gly-Lys- Arg (SEQ ID NO: 4) (M4PI)

In particular, primers with the following nucleotide sequences were prepared using an automatic DNA synthesizer:
Primer pBAM (SEQ ID NO: 25): 5′-GCAGGATCCA TGTTCGTTAA T-3 ′
Primer pHIN (SEQ ID NO: 26): 3′-ATAA CATTGATCAT TTTCGAAACG-5 ′
Primer psM1
Sinn Primer (SEQ ID NO: 29)
5′-GCTCCGGGTG ACGTTAAACG TGGCATCGTT GAACAA-3 ′
Antisense Primer (SEQ ID NO: 30)
3′-TGG GGCTTTTGGG CAGCGCGAGG CCCACTGCAA-s ′
Primer psM2
Sinn Primer (SEQ ID NO: 31)
5′-TACCCGGGTG ACGTTAAACG TGGCATCGTT GAACAA-3 ′
Antisense Primer (SEQ ID NO: 32)
3′-TGG GGCTTTTGGG CAGCGATGGG CCACTGAA-5 ′
Primer psM3
Sinn Primer (SEQ ID NO: 33)
5 ′ - CACCCGGGTG ACGTTAAACG TGGCATCGTT GAACAA- 3 ′
Antisense Primer (SEQ ID NO: 34)
3 ′ - TGG GGCTTTTGGG CAGCGGTGGG CCCACTGCAA- 5 ′
Primer psM4
Sinn Primer (SEQ ID NO: 35)
5 ′ -GGTCCGGGTA AACGTGGCAT CGTTGAACAA-3 ′
Antisense Primer (SEQ ID NO: 36)
3 ′ -TGGGGCT TTTGGGCAGC GCCAGGCCCA-5 ′

A DNA encoding miniproinsulin M1 was prepared as follows poses. A DNA fragment that contains the 5 'end region, the Miniproinsulin 1 ("sM1-A") was encoded by PCR under Use of the proinsulin gene prepared in Example 4 as Template and primer pBAM and antisense primer from psM1 manufactured. A DNA fragment that contains the 3'-end region, the Miniproinsulin 1 ("sM1-B") was also encoded by PCR using the proinsulin gene prepared in Example 4 as a template and the primer pHIN and the Sinn primer of psM1 manufactured. Equal amounts of sM1-A and sM1-B were mixed and recombined. A miniproinsulin Ml coding DNA was by PCR using the recombined DNA as a template and  the primers pBAM and pHIN were prepared.

The miniproinsulin M1 DNA was digested with BamHI and HindIII to to obtain a double-stranded DNA with cohesive ends that Detection sites for BamHI and HindIII at their 5′- and 3′- Contained ends.

The plasmid pT7-T1 prepared in Example 2 was treated with BamHI and HindIII digested and then using T4 DNA ligase the miniproinsulin M1 DNA treated with BamHI / HindIII, the had been obtained above. The resulting plasmid was called plasmid pT1-M1PI.

The DNAs encoding the miniproinsulins M2, M3 and M4 were prepared according to the same procedure as described above, except that each of the primers psM2, psM3 and psM4 was used instead of the primer psMl. Then the plasmids pT1-M2PI, pT1-M3PI and pT1-M4PI, which comprise the miniproinsulin M2, M3 and M4 DNAs, respectively, were also prepared according to the same method as above. The production methods of the plasmids pT1-M1PI, pT1-M2PI, pT1-M3PI and pT2-P4PI are shown in FIG. 3.

Example 8 Preparation of the plasmid pT2-MiPI

The plasmid pT7-T2 prepared in Example 3 was digested with BamHI and HindIII and then ligated using T4 DNA ligase to a BamHI / HindIII treated miniproinsulin M1 DNA. The resulting plasmid was called plasmid pT2-M1PI. The plasmids pT2-M2PI, pT2-M3PI and pT2-M4PI, which comprise miniproinsulin M2, M3 and M4 DNAs, respectively, were also prepared according to the same methods as above. The preparation methods of the plasmids pT2-M1PI, pT2-M2PI, pT2-M3PI and pT2-M4PI are shown in FIG. 4.

Example 9 Expression of the TNF miniproinsulin fusion protein

E. coli XL-1-Blue was prepared according to the Hanahan method as described in DNA Cloning, Volume 1, Ed. D.M. Glover, IRS Press, 109-135 (1985) with the plasmids pT1-M1PI, pT1-M2PI, pT1-M3PI, pT1- M4PI, pT2-M1PI, pT2-M2PI, pT2-M3PI, pT2-M4PI, pT1-hPI or pT2- hPI transformed. After that, they were on the solid medium formed ampicillin-resistant colonies and then selected 1 ml LB medium (10 g Bacto Trypton, 5 g Bacto yeast extract and 10 g NaCI pro per 1) containing 50 µg / ml ampicillin vaccinated.

The colonies were incubated at 37 ° C for 12 hours, and the resulting culture was zen at 8,000 × g for 2 minutes centrifuged, whereby E. coli cell pellets were obtained. Plasmids were carried out using the alkaline lysis method (Methods in Molecular Biology, Ed. Leonard G. Davis et al., Elsevier, S. 99-101 (1986)) separated from the pellets and 1 ug of the plasmid was dissolved in 50 ul TE buffer (pH 8.0). 0.1 µg of Plasmid solution was mixed with 2 μl of 10 × one-phor-all buffer (100 mM trisacetate, 100 mM magnesium acetate, 500 mM potassium acetate), 1 Unit NdeI and 5 units HindIII mixed and it was distilled water up to a final volume of 10 µl to the Given mixture. The solution was left at 37 ° C for 2 hours react, and then subjected them to a 1% agarose gel Electrophoresis to confirm insertion of a DNA fragment gene.

The plasmids which were confirmed to be the DNA fragment were used to express the expression host cell E.  

coli BL21 (DE3) according to the same procedure as above transform, and it became the ampicillin resistant colonies selected.

E. coli transformed with the plasmids pT1-M1PI or pT2-M2PI BL21 (DE3) cells, i.e. H. E. coli BL21 (DE3 + pT1-M2PI) and E. coli BL21 (DE3 + pT2-M2PI) were rearranged on December 29, 1994 KCCM-10059 or KCCM-10060 with the Korean Culture Center of microorganism deposited.

1 ml of an LB medium was mixed with the Ko Lions were vaccinated and the colonies were kept at 37 ° C for 12 hours bred. The culture was in 50 ml of a 50 µg / ml ampicillin containing LB medium, and once the optical Density of the culture at 600 nm was 0.1 to 0.4 IPTG (isopropylthiogalactopyranoside) to a final concentration of 1 mM added to the culture. The culture was for 4 hours kept at 37 ° C while stirring at 200 rpm, and then centrifuged at 8,000 rpm for 2 minutes, whereby E. coli cell pellets were obtained.

Example 10 Separation and purification of mini proinsulline

The E. coli cell pellets obtained in Example 8 were in 5 ml a 10 mM sodium phosphate buffer (pH 7.4) and then suspended subjected to ultrasound treatment to the cells to destroy. The cell lysate was 10 minutes at 4 ° C and 6,000 × g centrifuged to obtain pellets. The pellets were made with 10 times the volume of Triton X-100 buffer kept at 4 ° C (50mM Tris-HCl, pH 8.0, 10mM EDTA, 0.5% (v / v) Triton X-100, 100 mM NaCl) and then at 8,000 x g for 5 minutes centrifuged to separate the pellets. This washing and  Recovery steps were repeated twice.

The pellets were dissolved in 1 ml 0.1 M Tris-HCl (pH 8.9), the 6th M contained guanidine-HCl, and 50 mg of sodium sulfite and 25 mg of sodium tetrathionate was added to the solution. The resulting Mixture was reacted for 20 hours at room temperature and then centrifuged at 12,000 x g for 30 minutes, resulting in a supernatant liquid was obtained. The protruding one Liquid was placed in a Spectra Por # 3 dialysis membrane and then against distilled water at 4 ° C for 24 hours dialyzed. The resulting dialysate was kept for 30 minutes Centrifuged 10,000 × g, whereby precipitates were obtained. To the rainfall, urea and hydrochloric acid were up to Final concentrations of 8 M and 0.1 M were given. Then 5 mg Cyanogen bromide was added to the mixture and the resulting solution was Reacted for 20 hours at room temperature. The resulting Reaction mixture was carried out using an HPLC system (Hitachi, Japan) and a C-18 reverse phase column (Pharmacia) cleaned.

Example 11 Refolding of mini proinsulin and production of Insulin from it

The sulfonated miniproinsulin obtained in Example 10 was dried and then dissolved in 50 mM glycine buffer (pH 11.0) to a concentration of 53 µM. The resulting solution was cooled in ice and 50 mM 2-mercaptoethanol was added to a final concentration of 636 µM. The mixture was stirred at 4 ° C for 19 hours while the reaction vessel was kept sealed with a parafilm. 100 mM phosphoric acid was added to the reaction mixture to stop the reaction, and then the products were analyzed at 215 nm by HPLC. The result is shown in Figure 5, where (a), (b), (c), (d) and (e) represent the chromatograms of the correctly folded M1PI, M2PI, M3PI and M4PI and hPI. As shown in Fig. 5, each of the miniproinsulins of the present invention has a larger peak area, ie, a higher yield, than human proinsulin (hPI). The refolding yields of various miniproinsulins and human proinsulin are shown in Table 1.

Table 1

0.5 M sodium hydroxide was further added to a pH of 7.5 to 8.0 added to the reaction mixture. Trypsin and car boxypeptidase was added to the mixture in amounts such that the ratio of trypsin, carboxypeptidase B and Mi niproinsulin was set to 1: 5: 12,500. The mixture became Reacted at 16 ° C for 20 hours and then by adding 1 M hydrochloric acid adjusted to a pH of 2 to 3 in order to Stop reaction.

The resulting solutions were analyzed at 215 nm with an HPLC. The result is shown in Fig. 6, wherein (a), (b), (c), (d) and (e) represent the chromatograms of insulins by treating the refolded M1PI, M2PI, M3PI, M4PI and hPI with Trypsin and carboxypeptidase B have been produced. The result confirms that the miniproinsulins of the present invention give higher yields of insulin than human proinsulin.

Sequence list

Claims (16)

Human proinsulin derivative of formula (1): B-chain-ZA chain (I)
wherein: the A and B chains are human insulin chains, respectively;
Z is a peptide of the formula UUXPJ- (x ′) nUU;
U is an arginine or lysine residue;
X and X 'are independently an amino acid residue and (X') nn can be different or the same amino acid residue;
P is a proline residue;
J is a glycine, arginine or lysine residue; and
n is 0 or an integer from 1 to 5. 2. A human proinsulin derivative according to claim 1, wherein X is a Alanine, tyrosine, histidine or glycine residue; X ′ a Is aspartic acid, valine, arginine or glycine residue; and n is 2. 3. A human proinsulin derivative according to claim 1, wherein X is a Alanine residue; J is a glycine residue; and (X ′) n Aspa raginic acid-valine is. 4. A human proinsulin derivative according to claim 1, wherein X is a  Is tyrosine residue; J is a glycine residue; and (X ′) n As Paraginic acid valine. 5. A human proinsulin derivative according to claim 1, wherein X is a Is histidine residue; J is a glycine residue; and (X ′) n As Paraginic acid valine. 6. A human proinsulin derivative according to claim 1, wherein X is a Is glycine residue; J is an arginine residue; and (X ′) n arginine Is glycine. 7. A human proinsulin derivative according to claim 1, wherein X is a Is glycine residue; J is a glycine residue; and n is 0. 8. DNA that is a human proinsulin derivative according to one of the Claims 1 to 7 encoded. 9. Expression vector containing the DNA of claim 8. 10. Expression vector according to claim 9, which is the plasmid pT1-M1PI (KCCM-10059) or the plasmid pT2-M2PI (KCCM-10060). 11. Microorganism with a vector according to claim 9 or 10 has been transformed. 12. Microorganism according to claim 11, one with the plasmid pT1-MIPI (KCCM-10059) transformed E. coli BL21 (DE3). 13. Microorganism according to claim 11, which with the plasmid pT2- M2PI (KCCM-10060) transformed E. coli BL21 (DE3).   14. A process for the production of human insulin which includes the steps of: making a one encoding human proinsulin derivative of formula (I) DNA; Introducing the DNA into a vector, causing an Ex expression vector for the expression of human proinsu Linderivats is produced; Transform a host cell with the expression vector; Breeding the resulting Transformant under conditions that affect the expression of the enable human proinsulin derivative; Disconnect the human proinsulin derivative from culture and Manufacture of human insulin from human Proinsulin derivative by enzymatic hydrolysis.